WO1995034643A1 - Cellules d'e. coli, etc., a systeme suicidaire conditionnel - Google Patents

Cellules d'e. coli, etc., a systeme suicidaire conditionnel Download PDF

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Publication number
WO1995034643A1
WO1995034643A1 PCT/EP1995/002245 EP9502245W WO9534643A1 WO 1995034643 A1 WO1995034643 A1 WO 1995034643A1 EP 9502245 W EP9502245 W EP 9502245W WO 9534643 A1 WO9534643 A1 WO 9534643A1
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Prior art keywords
gene
cells
nuclease
promoter
plasmid
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PCT/EP1995/002245
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English (en)
Inventor
Wilfried Wackernagel
Michael G. Lorenz
Ingrid Ahrenholz
Manfred Jekel
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Wilfried Wackernagel
Lorenz Michael G
Ingrid Ahrenholz
Manfred Jekel
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Application filed by Wilfried Wackernagel, Lorenz Michael G, Ingrid Ahrenholz, Manfred Jekel filed Critical Wilfried Wackernagel
Priority to EP95923293A priority Critical patent/EP0717775A1/fr
Publication of WO1995034643A1 publication Critical patent/WO1995034643A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • GEMs genetically engineered microorganisms
  • the planned and the already performed applications of GEMs in agriculture, waste treatment, and production of certain raw materials rely on the release of great quantities of cells into the environment.
  • a main concern about releases of transgenic organisms comes from the uncertainty on how these organisms will behave in the environment (30) .
  • One aspect of this is the risk of unanticipated survival and reproduction in the environment with negative ecological effects.
  • Another problem is seen in the uncontrolled transfer of the genetically engineered DNA to other organisms in the environment.
  • conditional suicide systems for bacteria have been designed and constructed which cause the controlled death of the cells (for review see reference 20) .
  • Such suicide systems consist of a regulated killing gene which is expressed in response to specific factors in the environment or in closed systems.
  • the killing genes so far successfully used in suicide systems determined the synthesis of cell-lysing agents including membrane- destabilizing polypeptides (7, 14, 19), cell-destroying levane (23) and lysozyme (27) .
  • the genes were put under the control of promoters inducible by e.g.
  • An object of the invention is to provide a conditional suicide system by which cell death is accompanied by a degradation of intracellular DNA to acid soluble material.
  • the invention provides conditional suicide cells of Escherichia coli genetically engineered and comprising
  • nuclease gene which can be expressed in the cells by means of said promoter
  • the invention provides cells according to claim 1 or 2, characterized in that the cells can express altogether two or more nuclease genes.
  • the invention provides conditional suicide cells according to any of the preceeding claims, characterized by the gene for the nuclease of Serra tia marcescens deleted for the leader-coding sequence as said nuclease gene, where the nuclease gene is that of the wild type or a variant thereof which still kills cells of Escherichia coli and degrades intracellularly their DNA.
  • conditional suicide cells according to any of the preceeding claims, characterized by the lambda P L -promoter and/or the temperature sensitive repressor of phage lambda.
  • the invention provides plasmid pAH12 as plasmid according to claim 1(a) or claim 2(a), obtainable from plasmid pNuc by
  • step (b) cutting out the nuc gene together with the ribosome binding site and/or the T7PhilO promoter and the start codon from the plasmid resulting from step (a) and inserting said fragment downstream of the P L promoter of plasmid pSFl (after deletion of the ssb gene) and
  • F IG Construction of the containment plasmid pAH12 (for details refer to 'Results') .
  • nucleotide sequence of the leader peptide nuc coding sequence for the mature Serratia nuclease
  • ⁇ lO T7 ⁇ lO promoter
  • P L lambda L promoter
  • FIG. 2 Survival of E. coli TGE900 with the suicide plasmid pAH12 ( ⁇ ) or the vector pSFlE ( ⁇ ) after thermoinduction by incubation at 42 °C (0 to 40 min) . Viable counts were determined as described in 'Materials and Methods'.
  • the survival (N/NQ) is the viable count of the culture at the indicated times (N) divide -L by the viable count of the culture before induction (N Q ) .
  • the data are means of two (pSFlE) or three (pAH12) independent experiments.
  • FIG. 3 Intracellular DNA degradation in E. coli TGE900 pAH12 following thermoinduction by incubation at 42 °C (0 to 30 miri) .
  • the cells were labeled with [ 3 H]thymidine at 28 °C and treated as described in 'Materials and Methods'.
  • the TCA-insoluble radioactive material was determined in the thermoinduced culture ( ⁇ ) , in the culture thermoinduced in the presence of Cm (V) and in the culture kept at 28 °C throughout (O) .
  • Bacterial strains and plas ids Table 1 contains the description of bacterial strains and plasmids used in this study.
  • Escherichia coli AH1 was constructed by Pl transduction (18) using E. coli JC10289 pKY102 as donor of the recA deletion (12) .
  • Plasmid DNA was isolated by alkaline lysis (6) for treatment with restriction endonucleases and for transformations.
  • Serratia nuclease the extracellular nuclease of S. marcescens (in the following termed Serratia nuclease) during plasmid preparation the method of Birnboim and Doly (6) was modified. Before lysis the cells were washed in 0.5 ml 10 mM NaCl to remove extracellular nuclease. During the alkaline lysis incubation times were shortened to 5 min and all centrifugations were done at 4 °C.
  • the pellet was resuspended in 50 ⁇ l 10 mM Tris-HCl, pH 8.0, 100 mM EDTA, pH ⁇ 8.0. After addition of 25 ⁇ l 7.5 M NH 4 -acetate, pH 7.5 the mixture was incubated for 15 min at 70°C, for 10 min on ice and then centrifuged (12 min, 13.000 x g, 4°C) . The supernatant was extracted twice with phenol/chloroform/isoamylalcohol (25:24:1), once with chloroform/isoamylalcohol (24:1) and precipitated with ethanol.
  • the pellet was washed with ice-cold 70 % ethanol, dried and resuspended in 10 ⁇ l TE (10 mM Tris- HCl, pH 8.0, 1 mM EDTA pH 8.0) .
  • 10 ⁇ l TE 10 mM Tris- HCl, pH 8.0, 1 mM EDTA pH 8.0
  • the plasmid DNA was isolated with the Qiaprep-spin plasmid kit (Diagen, D ⁇ sseldorf, Germany) and extracted twice with phenol/chloroform/isoamylalcohol as described above.
  • E. coli was transformed by electroporation (Gene pul ⁇ er, Bio-Rad, Kunststoff, Germany; 25 ⁇ F, 12.5 kV cm - *1 , 200 Ohm)'.
  • DNA Intracellular DNA degradation.
  • DNA was labeled by growth of the cells in LB broth plus Ap (100 ⁇ g ml "1 ), 2'deoxyadenosine (250 ⁇ g ml -1 ) and [methyl- 3 H] thymidine (1.5 x 10 5 Bq ml" 1 ) for three generations at 28 °C.
  • Ap 100 ⁇ g ml "1
  • 2'deoxyadenosine 250 ⁇ g ml -1
  • [methyl- 3 H] thymidine 1.5 x 10 5 Bq ml" 1
  • the nuc gene coding for the Serratia nuclease determines a polypeptide of 266 amino acids of which the N-terminal 21 amino acids constitute a leader peptide (5) .
  • the leader peptide is removed during secretion which activates the mature nuclease (2, 5) .
  • the construction of a containment plasmid (pAH12) is described in Fig. 1.
  • the plasmid contains the nuclease gene of S. marcescens deleted for the leader-coding sequence under the control of the lambda L promoter.
  • E. coli TGE900 (8) which carries the gene for the thermosensitive lambda cI857 repressor in the chromosome the expression of the truncated gene for the leader- free Serratia nuclease is controlled by temperature.
  • the DNA fragment coding for the mature Serratia nuclease was cut out from the plasmid pNuc4 (2) by digestion with Eagl and BssHII. After mung bean nuclease digestion the fragment was joined to a start codon in the vector pET81F + (29) .
  • pET81F + was prepared for cloning by digestion with Ncol and BamHI and filling in of the resulting single-stranded ends of the vector with the Klenow fragment of DNA polymerase I of E. coli .
  • the resulting plasmid was termed pAHIO (Fig. 1) .
  • the truncated nuclease gene together with the ribosome-binding site and the start codon of p ⁇ T81F + was cloned downstream of the lambda P L promoter of the pBR322 derived plasmid pSFl (4) by blunt end ligation.
  • pAHIO was digested with S ⁇ pl , BamHI and mung bean nuclease and pSFl with EcoRI and mung bean nuclease.
  • the ligation mixture was transformed into E. coli TGE900.
  • thermoinduction of TGE900 with the vector pSFlE did not substantially affect growth during the 30 min induction period compared to the 28 °C culture.
  • the vector contained the ⁇ sb gene of E. coli coding for single-stranded DNA binding protein, the thermoinducted overproduction of this protein did not cause killing either.
  • E. coli TGE900 was transformed with plasmid DNA isolated from the four clones.
  • the transformant ⁇ showed the same growth in streak ⁇ on LB agar with Ap at 42 °C as the original surviving clones, suggesting that chromosomal mutations were not responsible for the survival of the clone ⁇ .
  • Gel electrophoretic analysis of the DNA of the four plasmid clones showed the same plasmid size as that of the wildtype plasmid pAH12.
  • Intracellular DNA degradation The extracellular Serratia nuclease is an endonuclease and degrades high molecular weight DNA to acid soluble material (21) . Previous studies had revealed that the nuclease introduces single- and double-strand breaks into duplex DNA which eventually leads to the breakdown of DNA into oligo- and mononucleotides (1) .
  • the cellular DNA was labeled with [ 3 H] thymidine and the fraction of T CA -insoluble radioactivity was determined after various incubation times. Following induction for 30 min at 42 °C DNA was degraded to acid soluble material for at least 2.5 h during which cell lysis did not occur. The data show that the Serratia nuclease without the leader peptide is active ih the cytoplasm of the cells. DNA degradation (and possibly RNA degradation) continued over a period of at least 3 h after derepression of the truncated nuc gene leading to the conversion of over 80 % of cellular DNA to acid soluble material. In cells grown at 28 °C all the time or thermoinduced at 42 °C with simultaneous addition of Cm (100 ⁇ g ml" 1 ) , there was little if any DNA degradation.
  • the suicide system presented here consists of the truncated nuc gene of Serratia marcescens (deletion of the sequence for a leader peptide) coding for a powerful DNase and' RNase cloned downstream of the lambda P L promoter and controlled by the thermosensitive lambda cI857 repressor.
  • cell killing correlated with DNA degradation, i.e. thermoinduction (30 min 42 °C) led to a minimum of cell survival and to extensive intracellular DNA breakdown.
  • thermoinduction (30 min 42 °C) led to a minimum of cell survival and to extensive intracellular DNA breakdown.
  • the expression of the nuclease gene is limited to the period before the coding sequences are destroyed by the enzyme.
  • the amount of enzyme produced following induction suffices possibly together with other cellular DNases to degrade the majority of intracellular DNA in the culture to acid soluble material within 3 h.
  • the intracellular milieu apparently does not only provide favorable conditions for the activity of the enzyme but also may contribute to the stability of the enzyme by providing a high concentration of proteins which was shown to stimulate the nuclease activity towards RNA and DNA in vitro and to stabilize the purified enzyme against thermal inactivation (1) .
  • the intracellular activity of the enzyme is notable, because the Serratia nuclease has two disulfide bonds, which are essential for the activity of the extracellular enzyme (3) , and the formation of which could be inhibited under the prevailing redox conditions in the intracellular milieu.
  • the efficiency of killing by the system (2 x 10 "5 ) is similar to or better than that of previously published' suicide systems not involving a nuclease. For example, efficiencies of 5 x 10 "2 (26), 10" 3 (23), 10" 5 to 10 "6 (7, 13) and 10 "8 by using two copies of the killing gene (13) were described. The fact that out of 25 examined survivors of a thermoinduction most were as sensitive as the initial strain and the rest was still highly sensitive suggests that the efficiency of the system is not very prone to mutational escape events. Knudsen and Karlstr ⁇ m (14) described a suicide system based on the relF gene of E. coli controlled by various lac promoters localized on plasmids.
  • the nucleolytic killing of cells is as ⁇ umed to prevent horizontal gene tran ⁇ fer effectively.
  • Cell ⁇ with degraded DNA cannot function a ⁇ donors in conjugation, tran ⁇ duction or transformation. Since cell lysi ⁇ did not occur following induction of killing, even large DNA fragments produced during initial ⁇ tage ⁇ of DNA degradation do not normally enter the environment where tran ⁇ formation of other cell ⁇ could occur. A l so, it seems unlikely that cells containing DNa ⁇ e (and degraded DNA) will pre ⁇ erve foreign DNA when acting as recipient.
  • the suicide system based on the controlled expression of a nucleotide sequence-independent nuclease can be' adopted for a variety of applications.
  • the nuclease killing gene could be put under the control of other regulators more relevant to the environment, such as those responding to starvation for specific substances.
  • the physiologically induced suicide of cells could substitute for killing by addition of chemicals.
  • cell lysis does not accompany killing, during industrial production of substances by GEMs the cell entity is preserved while the mass of DNA (and possibly RNA) is degraded leading to reduced efforts needed to remove nucleic acids from the product.
  • a chemical induction of the truncated nuclease gene expression could be desirable (such as IPTG) if high temperature regimes would have to be avoided due to a thermal sen ⁇ itivity of the product.
  • E. coli W TGE900 A(recA-srl) 306::Tn10, Tc R This work pET81F + Ap R , 2730 bp; expression vector with the 29
  • N/N Q The ⁇ urvival (N/N Q ) is the viable count of the culture at the indicated times (N) divided by the viable count of the culture before induction (N 0 ; for details, refer to 'Materials and Methods') .
  • the data are means of two independent experiments. TABLE 3. Thermoinduced killing of E. coli TGE900 pAH12 clone ⁇ which had survived a 42 °C treatment 3

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Abstract

Les risques potentiels liés à la libération intentionnelle ou non intentionnelle de micro-organismes obtenus par génie génétique, ont conduit à la construction de systèmes biologiques de confinement grâce auxquels les bactéries sont tuées dans un processus de suicide commandé. Dans les systèmes suicidaires préalablement publiés, la mort des cellules était causée par des protéines qui détruisaient la membrane ou paroi cellulaire. Ici, on décrit un système conditionnel de destruction de cellules, basé sur la dégradation intracellulaire de l'ADN cellulaire. On utilise le gène de la nucléase extracellulaire de Serratia marcescens, que l'on efface pour la séquence leader de codage, le gène tronqué étant placé sous la commande d'un promoteur lambda PL. Après induction thermique de la cassette du gène de nucléase dans Escherichia coli, la survivance cellulaire était tombée à 2 x 10-5, et plus de 80 % de l'ADN marqué de façon radioactive étaient convertis en matériau soluble dans l'acide pendant une période de 2,5 h, en l'absence de lyse cellulaire. Des cellules provenant de la majorité (84 %) des clones ayant survécu à la destruction par induction thermique, se sont révélées être aussi sensibles à une seconde induction thermique que la souche originale. Des cellules des autres clones ont démontré une cinétique de destruction en quelque sorte plus lente ou un niveau final de survivance légèrement supérieur. Le système suicidaire décrit combine la destruction régulée des cellules et l'avortement des procédés d'échange génique horizontal, par destruction de l'ADN disponible potentiellement d'une autre manière pour des conjugaisons, des transductions et des transformations génétiques.
PCT/EP1995/002245 1994-06-10 1995-06-09 Cellules d'e. coli, etc., a systeme suicidaire conditionnel WO1995034643A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024590A1 (fr) * 1997-11-06 1999-05-20 Commonwealth Scientific And Industrial Research Organisation Vecteur d'expression suicide pour souches de vaccin
WO1999050389A1 (fr) * 1998-03-30 1999-10-07 Metabolix, Inc. Souches microbiennes et procedes de production de biomateriaux
DE10160600A1 (de) * 2001-12-10 2003-06-26 Gl Biotech Gmbh Positives Selektionssystem zur Klonierung
US6964845B1 (en) 1996-08-21 2005-11-15 Werner Lubitz Thermostable phage lambda operator mutants for regulating gene expression

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987005932A1 (fr) * 1986-03-26 1987-10-08 Genexpress Aps Limitation biologique
EP0255755A2 (fr) * 1986-06-05 1988-02-10 Baylor College of Medicine Préparation de vaccin
WO1992014819A1 (fr) * 1991-02-26 1992-09-03 E.I. Du Pont De Nemours And Company Vecteur de selection positive pour un systeme de clonage de p1 bacteriophage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987005932A1 (fr) * 1986-03-26 1987-10-08 Genexpress Aps Limitation biologique
EP0255755A2 (fr) * 1986-06-05 1988-02-10 Baylor College of Medicine Préparation de vaccin
WO1992014819A1 (fr) * 1991-02-26 1992-09-03 E.I. Du Pont De Nemours And Company Vecteur de selection positive pour un systeme de clonage de p1 bacteriophage

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
G. RECORBET ET AL.: "Conditional suicide system of Escherichia coli released into soil thet uses the bacillus subtilis sacB gene", APPLIED MICROBIOL. AND BIOTECHNOL., vol. 59, no. 5, SPRINGER INTERNATIONAL, NEW YORK, US, pages 1361 - 1366 *
I. AHRENHOLTZ ET AL.: "A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA", APPLIED AND ENVIRONMENTAL MICROBIOL., vol. 60, no. 10, AM. SOC. MICROBIOL.,WASHINGTON, DC, US, pages 3746 - 3751 *
K. BIEDERMANN ET AL.: "Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli", CARLSBERG RES. COMMUN., vol. 54, SPRINGER-VERLAG, BERLIN, BRD, pages 17 - 27 *
S. MOLIN ET AL.: "Suicidal genetic elements an dtheir use in biological containment of bacteria", ANNUAL REVIEW OF MICROBIOL., vol. 47, ANNUAL REVIEWS INC. PALO ALTO, CALIFORNIA, US, pages 139 - 166 *
S.M. KNUDSEN AND O.H. KARLSTRÖM: "Development of efficient mechanisms for biological containment of bacteria", APPLIED AND ENVIRONMENTAL MICROBIOL., vol. 57, no. 1, AM. SOC. MICROBIOL.,WASHINGTON, DC, US, pages 85 - 92 *
T. SCHWEDER ET AL.: "An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells", APPLIED MICROBIOL. AND BIOTECHNOL., vol. 38, no. 1, SPRINGER INTERNATIONAL, NEW YORK, US, pages 91 - 93 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6964845B1 (en) 1996-08-21 2005-11-15 Werner Lubitz Thermostable phage lambda operator mutants for regulating gene expression
WO1999024590A1 (fr) * 1997-11-06 1999-05-20 Commonwealth Scientific And Industrial Research Organisation Vecteur d'expression suicide pour souches de vaccin
WO1999050389A1 (fr) * 1998-03-30 1999-10-07 Metabolix, Inc. Souches microbiennes et procedes de production de biomateriaux
US8728778B2 (en) 1998-03-30 2014-05-20 Metabolix, Inc. Microbial strains and processes for the manufacture of biomaterials
US8748139B2 (en) 1998-03-30 2014-06-10 Metabolix, Inc. Microbial strains and process for the manufacture of biomaterials
DE10160600A1 (de) * 2001-12-10 2003-06-26 Gl Biotech Gmbh Positives Selektionssystem zur Klonierung

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